A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol.

This study explored the role of fibulin-3 in osteosarcoma progression and the possible signaling pathway involved. Fibulin-3 mRNA and protein expression in normal tissue, benign fibrous dysplasia, osteosarcoma, osteosarcoma cell lines (HOS and U-2OS), the normal osteoblastic cell line hFOB, and different invasive subclones was evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC) and real time reverse transcriptase-polymerase chain reaction (real time qRT-PCR). To assess the role of fibulin-3 in the invasion and metastasis of osteosarcoma cells, lentiviral vectors with fibulin-3 small hairpin RNA (shRNA) and pLVX-fibulin-3 were constructed and used to infect the highly invasive and low invasive subclones. The effects of fibulin-3 knockdown and upregulation on the biological behavior of osteosarcoma cells were investigated by functional in vitro and in vivo assays. The results revealed that fibulin-3 expression was upregulated in osteosarcoma, and was positively correlated with low differentiation, lymph node metastasis, and poor prognosis. Fibulin-3 could promote osteosarcoma cell invasion and metastasis by inducing EMT and activating the Wnt/β-catenin signaling pathway. Collectively, our findings demonstrate that fibulin-3 is a promoter of osteosarcoma development and progression, and suggest a novel therapeutic target for future studies.

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The zinc-regulated transporters, iron-regulated transporter-like proteins (ZIPs), the natural resistance and macrophage proteins (NRAMP), the heavy metal ATPases (HMAs) and the metal tolerance or transporter proteins (MTPs) families are involved in cadmium (Cd) uptake, translocation and sequestration in plants. Mulberry (Morus L.), one of the most ecologically and economically important (as a food plant for silkworm production) genera of perennial trees, exhibits excellent potential for remediating Cd-contaminated soils. However, there is no detailed information about the genes involved in Cd2+ transport in mulberry. In this study, we identified 31 genes based on a genome-wide analysis of the Morus notabilis genome database. According to bioinformatics analysis, the four transporter gene families in Morus were distributed in each group of the phylogenetic tree, and the gene exon/intron structure and protein motif structure were similar among members of the same group. Subcellular localization software predicted that these transporters were mainly distributed in the plasma membrane and the vacuolar membrane, with members of the same group exhibiting similar subcellular locations. Most of the gene promoters contained abiotic stress-related cis-elements. The expression patterns of these genes in different organs were determined, and the patterns identified, allowing the categorization of these genes into four groups. Under low or high-Cd2+ concentrations (30 μM or 100 μM, respectively), the transcriptional regulation of the 31 genes in root, stem and leaf tissues of M. alba seedlings differed with regard to tissue and time of peak expression. Heterologous expression of MaNRAMP1, MaHMA3, MaZIP4, and MaIRT1 in Saccharomyces cerevisiae increased the sensitivity of yeast to Cd, suggested that these transporters had Cd transport activity. Subcellular localization experiment showed that the four transporters were localized to the plasma membrane of yeast and tobacco. These results provide the basis for further understanding of the Cd tolerance mechanism in Morus, which can be exploited in Cd phytoremediation.

Ethylene regulates plant abiotic stress responses and tolerances, and ethylene-insensitive3 (EIN3)/EIN3-like (EIL) proteins are the key components of ethylene signal transduction. Although the functions of EIN3/EIL proteins in response to abiotic stresses have been investigated in model plants, little is known in non-model plants, including mulberry (Morus L.), which is an economically important perennial woody plant. We functionally characterized a gene encoding an EIN3-like protein from mulberry, designated as MnEIL3. A quantitative real-time PCR analysis demonstrated that the expression of MnEIL3 could be induced in roots and shoot by salt and drought stresses. Arabidopsis overexpressing MnEIL3 exhibited an enhanced tolerance to salt and drought stresses. MnEIL3 overexpression in Arabidopsis significantly upregulated the transcript abundances of ethylene biosynthetic genes. Furthermore, MnEIL3 enhanced the activities of the MnACO1 and MnACS1 promoters, which respond to salt and drought stresses. Thus, MnEIL3 may play important roles in tolerance to abiotic stresses and the expression of ethylene biosynthetic genes.

Transfer printing based on switchable adhesive that heterogeneously integrates materials is essential to develop novel electronic systems, such as flexible electronics and micro LED displays. Here, we report a robust design of a thermal actuated switchable dry adhesive, which features a stiff sphere embedded in a thermally responsive shape memory polymer (SMP) substrate and encapsulated by an elastomeric membrane. This construct bypasses the unfavorable micro- and nano-fabrication processes and yields an adhesion switchability of over 1000 by combining the peel-rate dependent effect of the elastomeric membrane and the thermal actuation of the sub-surface embedded stiff sphere. Experimental and numerical studies reveal the underlying thermal actuated mechanism and provide insights into the design and operation of the switchable adhesive. Demonstrations of this concept in stamps for transfer printing of fragile objects, such as silicon wafers, silicon chips, and inorganic micro-LED chips, onto challenging non-adhesive surfaces illustrate its potential in heterogeneous material integration applications, such as flexible electronics manufacturing and deterministic assembly.

Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1–methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening.

Visfatin is considered to be a biomarker in various types of cancers. However, no evidence has been reported for the direct effect of visfatin on osteosarcoma cell metastasis. The aims of the present study were to investigate the influence of visfatin on the migration and invasion of osteosarcoma cells and clarify the underlying mechanism. The expression levels of epithelial-mesenchymal transition (EMT) markers, as well as the transcriptional factor Snail-1, were first detected at both the protein and mRNA levels in U2OS osteosarcoma cells after stimulation of visfatin. Then the expression of NF-κB (p65) was detected by western blot analysis, and siRNA of Snail-1 and inhibitor of NF-κB were used to investigate the effect of visfatin. Finally, migration and invasion of the cells were detected respectively by scratch wound healing and transwell assays. Visfatin downregulated E-cadherin and upregulated N-cadherin in concentration- and time-dependent manners at the protein and mRNA levels. The expression of Snail-1 was also upregulated. Moreover, visfatin also promoted the nuclear translocation of the NF-κB pathway. Administration of siRNA of Snail-1 and the inhibitor BAY11-7082 validated the roles of Snail-1 and NF-κB in the visfatin-induced regulation of EMT markers. Migration and invasion of U2OS osteosarcoma cells were promoted following the application of visfatin. These results demonstrated that visfatin enhances the migration and invasion of osteosarcoma cells via the NF-κB/Snail-1/EMT pathway.